The Prostate Specific Membrane Antigen (PSMA) is a 750-amino acid type II transmembrane protein. PSMA is expressed by prostatic epithelial cells and extraprostatic expression has been detected in the brain, kidney, salivary gland and duodenum. (See e.g. Renneberg et al. (1999) Urol. Res. 27(1):23-7; Troyer et al. (1995) Int. J. Cancer 62(5):552-8; Israel et al. (1994) Cancer Res. 54(7):1807-11; Israel et al. (1993) Cancer Res. 53(2):227-30). PSMA is a carboxypeptidase which cleaves N-acetyl-asp-glu. PSMA has three domains: a 19-amino acid cytoplasmic domain, a 24-amino acid transmembrane domain, and a 707-amino acid extracellular domain. A monoclonal antibody specific to the cytoplasmic domain, 7E11.C5, has been adapted for in vivo imaging of prostatic cancer through radiolabeling with indium-111. (Elgamal et al. (1998) Prostate 37(4):261-9; Lamb and Faulds (1998) Drugs Aging 12(4):293-304).
Since its discovery in 1987 (Horoszewicz et al. (1987) Anticancer Res. 7:927-35), PSMA has been considered an excellent prostate tumor cell marker. PSMA expression is primarily prostate specific, with barely detectable levels seen in the brain, salivary glands, and small intestine (Israeli et al. (1994) Cancer Res. 54:1807-11). Additionally, PSMA expression is high in malignant prostate cells, with the highest expression in androgen resistant cells due to negative regulation by androgens (Wright et al. (1996) Urology 48:326-34). Furthermore, PSMA is alternatively spliced, where normal prostate cells predominantly express a cytosolic form named PSM′ and malignant cells express the characteristic full-length membrane bound form (Su et al. (1995) Cancer Res. 55:1441-3). This full-length PSMA is a type II membrane glycoprotein, in which the majority of the protein is extracellular and available as a target for diagnostic and therapeutic agents. These properties have made PSMA an ideal target for prostate cancer immunotherapy (Murphy et al. (1999) Prostate 39:54-9); monoclonal antibody imaging (Sodee et al. (1998) Prostate 37:140-8); and therapy (McDevitt et al. (2000) Cancer Res. 60:6095-100). The first anti-PSMA antibody was quickly modified into an imaging agent (Lopes et al. (1990) Cancer Res. 50:6423-6429), which is currently used clinically to diagnose metastatic prostate tumors. Additionally, PSMA is expressed by neovascular endothelial cells in a variety of cancers (Chang et al. (1999) Clin. Cancer Res. 5:2674-81; Liu et al. (1997) Cancer Res. 57:3629-34), making it a candidate target for tumor vascular imaging and anti-angiogenesis therapy.
An aptamer that recognizes PSMA's extracellular domain has potential utility as a therapeutic entity, via inhibition of PSMA enzymatic activity, as an in vivo imaging agent, and additionally as a targeting agent for therapeutic delivery of cytotoxic chemicals and radionuclides. The use of proteins as drugs and reagents is often limited by the activity of proteases, the size of the protein, transport and the ability of an organism to make antibodies against that protein. Many of these limitations can be circumvented by the use of aptamers, made of synthesized RNA, that are stabilized against nuclease activity. Relative to antibodies, aptamers are small (7-20 kDa), clear very rapidly from blood, and are chemically synthesized. Rapid blood clearance is important for in vivo diagnostic imaging, where blood levels are a primary determinant of background that obscures an image. Rapid blood clearance may also be important in therapy, where blood levels may contribute to toxicity. SELEX technology allows rapid aptamer isolation, and chemical synthesis enables facile and site-specific conjugation of aptamers to a variety of inert and bioactive molecules. An aptamer to PSMA would therefore be useful for tumor therapy or in vivo or ex vivo diagnostic imaging and/or for delivering a variety of therapeutic agents complexed with the PSMA nucleic acid ligand for treatment of disease conditions in which PSMA is expressed.
The development of the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process has provided a new alternative, nuclease-resistant oligonucleotides that can be selected to bind tightly and specifically to almost any ligand. (Tuerk and Gold (1990) Science 249:505-10; Ellington and Szostak (1990) Nature 346:818-22; Lin et al. (1994) Nucleic Acids Res. 22:5229-34; Gold (1995) J. Biol. Chem. 270:13581-4); for example: organic dyes, antibiotics, amino acids, and cells (Ellington and Szostak (1990) Nature 346:818-22; Wang and Rando (1995) Chem. Biol. 2:281-90; Connell et al. (1993) Biochemistry 32:5497-502; Morris et al. (1998) Proc. Natl Acad. Sci. USA 95:2902-7). These synthetic oligonucleotide sequences, termed “RNA aptamers,” have been made to bind over 100 target ligands and are emerging as a new class of molecules that contest antibodies in therapeutics, imaging, and diagnostics (Hicke and Stephens (2000) J. Clin. Invest. 106:923-8; Jayasena (1999) Clin. Chem. 45:1628-50).
The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands,” each of which is specifically incorporated herein by reference in its entirety. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. Nos. 5,763,177 and 6,011,577, both entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptide, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement,” describes oligonucleotides containing various 2′ modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,” respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. Pat. No. 6,011,020, entitled “Nucleic Acid Ligand Complexes.” Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
Since the first discovery of RNA aptamers as ligand binding agents (Tuerk and Gold (1990) Science 249:505-10; Ellington and Szostak (1990) Nature 346:818-22), an enormous diversity of target molecules have been identified (Famulok et al. (2000) Acc. Chem. Res. 33:591-9). The diversity of structures employed by an aptamer library allows tight binding RNA ligands from targets as simple as a single amino acid (Connell et al. (1993) Biochemistry 32:5497-502), to complex targets such as red blood cells (Morris et al. (1998) Proc. Natl Acad. Sci. USA 95:2902-7). Despite the success of this technique, however, there are no reported RNA aptamers to membrane bound tumor antigens. Therefore, the possibility of identifying and producing nuclease stable RNA aptamers that bind to and inhibit the enzymatic activity of the well-known prostate tumor cell surface antigen, PSMA was explored.
It is an object of the present invention to provide methods that can be used to identify nucleic acid ligands that bind with high specificity and affinity to PSMA.
It is a further object of the present invention to obtain nucleic acid ligands to PSMA that inhibit the activity of PSMA when bound.
It is a further object of the present invention to provide a complex for use in in vivo or ex vivo diagnostics comprising one or more PSMA nucleic acid ligands and one or more markers.
It is a further object of this invention to provide a method for delivering therapeutic agents for the treatment or prophylaxis of disease conditions in which PSMA is expressed.